Hello,
I extracted mouse liver tissue using triton-x-1% with several different protease and phosphatase inhibitors. I did the same thing with NIH3T3 cells and AML12 cells. When I detect actin, everything works just fine.
When I do the procedure to detect my protein of interest, APOE, the protein has several non-specific bands and most important has a strong non-specific band right where my protein is, all from the secondary antibody.
Also I do not see the APOE in the AML12 cells at all and I believe it is supposed to be expressed in AML12 cells.
Primary APOE Antibody: SantaCruz sc-13521
Secondary Antibody: Invitrogen A16072
NOTE: It is expected that the second liver band was supposed to be slightly less so I thought I was accurately detecting my protein.
Hi Benjamin,
You may do one or a combination of the following.
1. Increase the concentration of blocking agent such as if you are using 3% BSA then use 5% BSA.
2. Increase the time of blocking, such as if you are doing 30 min blocking then do 1h blocking.
3. Dilute the antibodies (primary/secondary) in 5% BSA(PBS+0.1%TBST).
4. If you don't get expected result in all the above then change the secondary antibody and use a different source altogether.
Good luck
The Invitrogen A16072 is a Goat anti-Mouse IgG (H+L) secondary antibody. So, if protein samples extracted from mouse liver tissue contain mouse IgGs, it is probably that the heave chain(H) and light chain(L) of mouse IgG may be detected, with strong siganls, as shown in 'Secondary only' lanes. Change to secondary antibodies that do not react with SDS-denatured mouse IgG may improve the detection.
Hi,
Multiple reasons could be possible for this, but based on your results I would suggest you to run a secondary antibody control (without Primary antibody) and try decreasing the concentration of primary antibody, I think it will surely give you a clear picture why your experiment is failing.
Second reason, could be the presence of different splice variants sharing same epitopes, and APOE is known to have several variants, and if reason is second one then it would be tricky and I would suggest you to perform BLAST search before starting the experiments.
Hope it works.
What concentration of secondary are you using? You may want to try reducing your secondary by 10-fold. Also, you can try a casein block instead of BSA and block for longer (try 40-60 minutes).
It depends on the method you chose. Most times, each of the methods available specify a particular blocking solutions. If your secondary antibody is to be tagged with HRP it means peroxidase solution may serve well for your blocking.
Blocking means prevention of nonspecific site to be falsely reacted with your Ab of interest thereby preventing false positive result or increased signal unnecessarily.
From Ling's answer I gather that you are using an anti mouse secondary antibody. That would mean that the primary antibody source is mouse.
However, it is not possible that mouse will generate antibody to its own antigen.
Thus you will need to change bot your primary and secondary antibody.